Abstract Understanding the exact cell-type composition in brain regions is a fundamental step when trying to integrate physiological, behavioral, neurochemical and molecular data. At present, although major categories of cell- types present in cortex have been defined through a handful of specific markers, the different subtypes within these categories, as well as their location and connectivity are far from understood. DNA methylation (mC) is a stable covalent modification that persists in post-mitotic cells throughout their lifetime, defining their cellular identity. Methylation patterns are cell type specific, differentiating the major types of cells i.e., neurons and glia, in the rodent and human cortex, as well as differentiating neuronal types in mouse cortex. However, little is known about what identifies the populations of neurons belonging specific categories within a brain region e.g. pyramidal neurons in different cortical layers or areas of frontal cortex. This project proposes to develop the technology necessary to address the challenge of producing single-neuron mC maps in a cortical layer-specific manner using human postmortem frontal-cortex tissue. Expansion of this methodology will ultimately lead to a thorough classification of all neuronal types in the human brain.